The long-term goal of this work is to determine the molecular and cellular mechanisms required for sperm-egg fusion during fertilization. This proposal addresses how a sperm secretory vesicle (acrosome) forms properly so that it can fuse with and create a fertilization-competent cell surface on the spermatozoon. Sperm secretory vesicles acquire an acidic internal pH during their maturation due to vacuolar (V-) ATPase activity, and this occurs in many animals, including humans. However, the mechanisms that regulate V-ATPase activity in sperm secretory vesicles are not understood, mostly because a spermatogenesis system suitable for its analysis had not been identified. Our preliminary data show that Caenorhabditis elegans spermatogenesis is ideally suited for analyzing the V-ATPase role and regulation during biogenesis and function of sperm secretory vesicles. In particular, mutations in the spe-5 gene alter a V-ATPase subunit that is spermatogenesis-specific, so acidification of sperm secretory vesicles can be studied in the absence of somatic V-ATPase defects, which are usually lethal. The spe-16 gene encodes a spermatogenesis-specific ubiquitin E3 ligase and how it participates in developmentally controlled acidification of sperm secretory vesicles will be determined. The focused objective of this proposal is to determine how the spe-5 and spe-16 encoded proteins cooperate to regulate internal pH in sperm secretory vesicles and what happens when this regulation is disrupted. We use three specific aims to test the overall hypothesis that the internal pH of sperm secretory vesicles must fall in order for this organelle to fuse with the plasma membrane. Specific aim #1 is to identify the role of the V-ATPase during sperm secretory vesicle maturation. Specific aim #2 is to identify how absence of V-ATPase function affects sperm motility and function during fertilization. Specific aim #3 is to identify why secretory vesicles fail to acidify in spe-16 loss of function mutant sperm. The unusual nature of the spe-16 mutant phenotype ensures it utilizes a previously undescribed way of regulating an animal V-ATPase. Ubiquitin is conjugated to proteins in normal human sperm, including those in the acrosome, and ubiquitination defects occur in infertile men;the role of ubiquitination is not understood in either case. Defects in ubiquitin biology are associated with various cancers, neurodegenerative diseases and many other disease processes. In addition to its role during spermatogenesis, V-ATPase function is defective in diseases that include renal Fanconi syndrome and the bone diseases, infantile ostepetrosis and osteoporosis. This proposal is focused on how sperm develop the ability to fertilize an egg. This is a problem of significant public health impact because ~15% of couples desiring children are infertile and many cases are due to defective sperm. The processes we study, in addition to functioning in sperm, also play a role in proper kidney function and bone formation.